Diabetes is caused by the loss or dysfunction of insulin-secreting β-cells in the pancreas. β-cells reduce their mass and lose insulin-producing ability in vitro, likely due to insufficient cell-cell and cell-extracellular matrix (ECM) interactions as β-cells lose their native microenvironment. Herein, we built an ex-vivo cell microenvironment by culturing primary β-cells in direct contact with ‘synthetic neighbors', cell-sized soft polymer microbeads that were modified with cell-cell signaling factors as well as components from pancreatic-tissue-specific ECMs. This biomimetic 3D microenvironment was able to promote native cell-cell and cell-ECM interactions. We obtained sustained maintenance of β-cell function in vitro enhanced cell viability from the few days usually observed in 2D culture to periods exceeding three weeks, with enhanced β-cell stability and insulin production. Our approach can be extended to create a general 3D culture platform for other cell types.